brain avm models and novel therapeutic targets · novel therapeutic targets 2nd ucsf stroke and...

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CCR UCSF center for cerebrovascular research

Brain AVM Models and Novel Therapeutic Targets

2nd UCSF Stroke and Aneurysm Update CMESaturday September 6, 2014

Hua Su, MD. Professor

Center for Cerebrovascular ResearchDepartment of Anesthesia and Perioperative Care

University of California, San [email protected]


I have nothing to disclose.


Brain Arteriovenous Malformations (AVMs)

•Tangle of abnormal blood vessels (nidus) –Arteriovenous shunting–No intranidal capillary bed–Range of vessel types

• Located randomly throughout brain• Cause of hemorrhagic stroke


Current TreatmentsSurgery, embolization and radiosurgery

No specific medical therapy for brain AVM

Dea

th o

r S

toke

(%

)

Months

Invasive therapy (n=114)

HR=0.27 (95% CI: 0.14-0.54)

Medical management (n=109)

The goals of specific medical treatments are:

1. Stabilize vessel wall-reduce spontaneous intracranial hemorrhage and hemorrhagic

stroke2. Reduce brain AVM grow or regrow after invasive treatment3. Reduce AVM volume

-surgical resection easier-reduce risk of invasive procedures

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Identify Specific Targets

-Analyzing surgical specimens-Modeling brain AVM in animals


Macrophage & Leukocytes

smooth muscle

VEGF

VEGF-R

MMP-9

Tie-2

Imbalance in Angiopoietin 1 & 2

astrocyte

Hashimoto, Neurosurgery 54: 410, 2004

Shenkar, Neurosurgery 52: 465, 2003 Kilic, Neurosurgery 57: 997, 2005Sure, Neurosurgery 55: 663, 2004Sonstein; J Neurosurg 85:838, 1996ZhuGe, Q. et al. Brain 2009

Murphy, PA. Laboratory Investigation 2009

Tissue assays of surgical specimens: “angiogenesis run amok”

“a healing wound”

endothelium

aVB3Ki-67

HIF-1α

Notch

Notch


Are brain AVMs heritable?

• Familial

– Hereditary Hemorrhagic Telangiectasias (HHT)

– RASA1 (p120 RasGAP, is a Ras GTPase–activating protein) capillary malformation-AVM

• Eerola, Am J Hum Genet 73: 1240, 2003

– Non-HHT• 53 patients in 25 families

– van Beijnum, et al, JNNP 78: 1213, 2007

– Inoue, et al, Stroke 38: 1368, 2007

• Sporadic 95-98% no family history


• Autosomal dominant disorder

• Mucocutaneous telangiectasia

• AVMs in Liver, Lung and Brain

• 80% of cases have functional heploinsufficiency of

Endoglin (HHT1) or ALK1 (HHT2)

Hereditary Hemorrhagic Telangiectasia (HHT)Rendu-Osler-Weber Syndrome

Liver AVMLung AVM Brain AVMs

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Eng+/- and Alk1+/- mice –minimal brain phenotype

Satomi, et al, Stroke 2003;34:783

Corrosion casting and SEM revealed AVMs in 3/10 mice

Srinivasan, et al, Hum Mol Genet 12: 473, 2003

In >47 mice, one Alk1+/- with dilated cerebellar vessel


AdCre – Regional Conditional Deletion of Alk1

loxp

loxp

CMV Promoter Cre recombinase

Promoter

Promoter

loxp

AdCre

Exons 4, 5, 6Exo

n 3

Exo

n 7

Exo

n 3

Alk 1 gene

Exons 4,5,6 are deleted from Alk1 genome

Exo

n 7


Alk1 Regional Conditional Deletion Plus VEGF Stimulation Results in Brain AVM

AdCre + AAV-VEGF

8 wks

Alk1 -/-

Angiogenesis

Walker et al. Ann Neurology, 2011 CCR UCSF center for cerebrovascular research

Alk1+/+/VEGF

Alk1-/- onlyAlk1-/- /VEGF

VEGF Stimulation is Necessary for Brain AVM Formation

Alk1+/+/VEGF

Walker et al. Ann Neurology, 2011

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Chen et al. Translational Stroke Research, 2014

Adult onset AVM models

Choi et al., PLOS One, 2014

CCR UCSF center for cerebrovascular researchChen et al, Stroke, 2014

Some Models have AVM in Other Organs

skin


Macrophage Infiltration

Chen et al. ATVB, 2013 CCR UCSF center for cerebrovascular research

AVM vessels have less smooth muscle cell coverage

Chen et al. ATVB, 2013

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AVM vessels have less pericyte coverage

Chen et al. ATVB, 2013 CCR UCSF center for cerebrovascular research

Microhemorrhage

Chen et al. ATVB, 2013


ALK1 Knockdown Attenuates the Upregulation of PDGFB in HBMEC in Response to VEGF Stimulation

HBMEC (human brain microvascular endothelial cell) were transfected with control shRNA or shRNA . Cells with >70% reduction of Alk1 gene expression were cultured for 18 h in the presence or absence of VEGF (0, 10, 50, and 100 ng/ml). qRT-PCR was performed for Alk1(A) and Pdgfb (B). All data are shown as mean and SD. *p<0.05 vs. control.

B

0

1

2

3

4

5

Pd

gfb

mR

NA

Fol

d C

hang

e

ControlshAlk1

VEGF 0 10 50 100(ng /ml)

**

*

0

0.5

1

1.5

2

2.5

3

Alk

1m

RN

A F

old

Cha

nge

ControlshAlk1

A

VEGF 0 10 50 100(ng /ml)

* * * *


ALK1 knockdown in HBMEC impairs the pericyte recruitment

20 40 60

VEGF + shAlk1

shAlk1

VEGF

Control

Average Pericyte Distance µm

A B

**

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50 µm

Gene Mutation in Bone Marrow Transmits the Phenotype


Reduction of Gene Mutant Endothelial Cell Reduced GI Hemorrhage and Mortality


BMDC/Monocyte

Inflammation

Angiogenesis

Therapies

Anti-angiogenesis(bevacizumab, sFLT)

BM or monocytetransfusion

Anti-inflammation(tetracycline class)

Important Factors in AVM Pathogenesis

Impaired mural cell recruitment

Improve vascular integrity(Thalidomide,Lenalidomide)


Anti-AngiogenesisBevacizumab reverse brain AVM phenotype

Walker et al. Stroke, 2012

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Anti-AngiogenesisStereotactic Injection of AAV2-sFLT Inhibited Brain AVM Formation

AAV2-EV

AAV2-sFLT02

1. Block VEGF that is used for model induction2. Invasive intra-brain injection


Developmental onset

1. SM22α-Cre mediated Eng deletion 2. 95% mice have brain AVM at five weeks of age3. Brain AVM in this model was developed spontaneously without local

angiogenic stimulation4. About 30% mice died between 3 and 6 weeks

Choi et al., PLOS One, 2014


number motality paralyzed final mice number

AAV9-GFP 9 3 1 5

AAV9-sFLT 8 2 6

Intravenous Injection of AAV9-sFLT ReversedBrain AVM Phenotype

SM22αCre;Engf/f mice

1. 1X1011 vg AAV9-sFLT IV to 5 weeks old mice.

2. Samples were collected4 weeks later

1. AAV-sFLT reverse brain AVM phenotype2. Systemicdelivery of AAV-sFLT is feasible3. AAV-sFLT is effect on spontaneous

developed bran AVM


Lebrin, et al, Nat Med 16: 420, 2010

Increase PDGFB

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Increase PDGFB Thalidomide Treatment Reduced the Number of Abnormal Vessels


Increase PDGFB Thalidomide Treatment Reduced Microhemorrhage


Summary

1. Invasive therapies are associated with considerable risks2. No specific medical therapy is available3. The concept for the treatment of brain AVM is to

stabilize vascular tissue and thereby decrease the risk of spontaneous ICH.

4. Novel therapeutic approaches: A. Anti-inflammationB. Anti-angiogenesisC. Improve vascular integrityD. Correct gene mutation in BM monocyte/progenitors


Thank You

William L. Young Mervyn MazeHelen KimLudmila PawlikowskaMichael T. LawtonCharles E. McCulloch

Funding:NIH AHAMichael Ryan Zodda Foundation

Espen WalkerWanqiu ChanEunjung ChoiFanxia ShenYi GuoLei MaoMarine CamusMamta WankhedeZhengyi HanYue HeCameron McDougallLiang WangLei ZhanShuai KangWan ZhuRui ZhangDingquan Zou

brain avm models and novel therapeutic targets · novel therapeutic targets 2nd ucsf stroke and...

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